The present invention relates to a new method of use for creatine compounds such as creatine kinase inhibitors and, more particularly, cyclocreatine, to block intracellular signals of the thrombin receptor Protease Activated Receptor-1, to thereby inhibit thrombin-induced cytoskeletal reorganization, platelet aggregation and endothelial cell contraction.
Platelets are discoid cells found in large numbers in blood, which are important for blood coagulation and hemostasis. Upon activation by various stimuli like thrombin, thromboxane A2 and ADP, platelets change into a spheroid shape with filopodia, degranulate and aggregate. Platelet activation is important for hemostasis and underlies various pathological conditions such as unstable angina pectoris, myocardial infarction, stroke, and coagulopathies. One of the physiological agents that activate platelets is thrombin, a serine protease. Thrombin mediates its action through the activation of protease activated receptors (PARs). Cytoskeletal rearrangement and shape changes precede platelet aggregation following thrombin receptor activation. Thrombin mediates vascular permeability, morphological changes in neurons and astrocytes through activation of PAR-1. Two thrombin receptors, high affinity PAR-1 and low affinity PAR-4, have been identified on human platelets. Low concentrations (1 nM) of thrombin activates PAR-1 in human platelets, while complete activation of platelets require higher amounts ( greater than 10 nM) of thrombin and activation of PAR-4.
Endothelium forms a barrier between blood and underlying tissues in the vascular system. Vascular endothelium actively participates in regulating vascular tone, inflammation, haemostasis and thrombosis. Endothelial cells express and release a repertoire of molecules that regulate vascular as well as endothelial function. During vascular injury and in inflammation thrombin released from the blood activates thrombin receptors (PARs) expressed on endothelial surface. Thrombin and thrombin receptors regulate various endothelial functions and play a pivotal role in vascular development and in the response of endothelial cells to vascular injury. Thrombin released during vascular injury has diverse effects on endothelial cells. Thrombin induces cytoskeletal changes resulting in cell rounding. Contraction of endothelial cells leads to increased permeability and compromises in the endothelial barrier. Thrombin also causes endothelial cells to proliferate during vascular injury. Endothelial cells release various vasoactive substances during wound healing in the presence of thrombin. High affinity thrombin receptor, PAR-4, is not expressed on endothelial cells and, therefore, most of the thrombin responses in endothelial cells are PAR-1 mediated.
PAR-1 mediates the cellular responses to thrombin during blood coagulation, cell proliferation, vascular permeability changes, tumor metastasis, and nervous system injury. PAR-1 is a seven-transmembrane G protein-coupled receptor with a novel activation mechanism. Proteolysis at a thrombin cleavage site in the extracellular amino terminus exposes a new amino terminus containing the peptide ligand SFLLRN, which binds intramolecularly to initiate intracellular signals. Although originally detected in platelets, endothelial cells, and fibroblasts, PAR-1 is also expressed in the nervous system in a developmentally regulated manner and by specific subpopulations of neurons and astrocytes that are especially vulnerable to neurodegeneration and ischemic injury.
In most cells expressing PAR-1, activation of the receptor transmits signals to the actin cytoskeleton that profoundly alter cell shape. Platelets, for example, convert from a spherical to disk shape and extend filopodia, endothelial cells contract, neurons retract axons, and astrocytes resorb processes and flatten their cell bodies. These signals also regulate changes in actin-related cell motility observed in neurons, fibroblasts, and tumor cells. The morphological response is mediated by a key signaling pathway that uses serine/threonine kinases, G12/13, RhoA, and myosin light chain kinase; actomyosin contraction generates tension through the formation of stress fibers and focal adhesions. There are several points along this signal transduction pathway at which high-energy phosphate in the form of ATP is required. Only phosphorylated myosin is capable of interacting with actin filaments, and actin polymerization requires binding to ATP. Depletion of total cellular ATP or the application of drugs that block the ATP binding sites of serine-threonine kinases, for example, inhibits PAR-1-mediated shape changes. Activation of G12/13 requires phosphorylation and a ready supply of GTP. Thus, the actomyosin contraction underlying cellular shape changes is an active, energy-consuming process where localized ATP homeostasis is critical.
Creatine kinase enzymes regulate ATP homeostasis in subcellular compartments by the transfer phosphates between creatine and adenine nucleotides. The mitochondrial isoform generates creatine phosphate, which is shuttled to cytosolic isoforms strategically localized to specific subcellular regions where they provide ATP at sites of high and fluctuating energy demand. In sea urchin sperm, for example, mitochondrial creatine kinase is restricted to sperm heads whereas cytosolic creatine kinase is expressed in the sperm tail, and specifically inhibiting ATP generation by the cytosolic creatine kinase results in defective tail movements and sperm motility. The cytosolic muscle isoform localizes along the M-line to the myosin heads where it provides ATP during actomyosin contraction. The cytosolic brain isoform (CKB) and its insect homolog arginine kinase localize to the membranes of neurons and astrocytes and are concentrated along axons and moving lamellipodial edges of migrating glia, although the precise function of CKB is not known.
The creatine kinase/creatine phosphate system is an energy generating system operative predominantly in the brain, muscle, heart, retina, adipose tissue and the kidney. Wallimann et al., Biochem. J., 281: 21-401 (1992). The components of the system include the enzyme creatine kinase (CK), the substrates creatine, creatine phosphate (CrP), ATP, ADP, and the creatine transporter. Creatine kinase is a phosphotransferase, which catalyzes reversibly at localized intracellular sites the transfer of a phosphoryl group from creatine phosphate to ADP to generate ATP which is the main source of energy in the cell. CK plays a key role in the energy homeostasis of cells with intermittently high, fluctuating energy requirements, like neurons, and photoreceptor ,and muscle cells. CK is expressed in a tissue specific manner: CK-M (muscle form) and CK-B (brain form). CK is localized in discrete cellular compartments coupled functionally to sites of energy production (glycolysis and mitochondria) or energy consumption (acto-myosin ATPase and Ca++-ATPase).
Some of the functions associated with CK/CrP system include efficient regeneration of energy in the form of ATP in cells with fluctuating and high energy demand, energy transport to different parts of the cell, phosphoryl transfer activity, ion transport regulation, and involvement in signal transduction pathways.
The substrate creatine is a compound that is naturally occurring and is found in mammalian brain, skeletal muscle, retina, adipose tissue and the heart. The phosphorylated form, creatine phosphate, is also found in the same organs and is the product of the creatine kinase reaction. Both compounds can be easily synthesized and are non-toxic to humans and animals. A series of creatine analogs, including cyclocreatine, homo cyclocreatine and xcex2-guanidino propionic acid, have been synthesized and shown to be efficacious for combating viral infections (U.S. Pat. No. 5,321,030); for inhibiting growth, transformation, or metastasis of mammalian cells (U.S. Pat. Nos. 5,324,731 and 5,676,978); for treatment of obesity (U.S. Pat. No. 5,998,457); and for the prevention and treatment of glucose metabolic disorders (U.S. Pat. No. 6,075,031), which are expressly incorporated herein by reference in their entireties. The use of cyclocreatine for restoring functionality in muscle tissue is disclosed in U.S. Pat. No. 5,091,404. PCT publication No. WO 94/16687 describes a method for inhibiting the growth of several tumors using creatine and related compounds. No prior work has established a direct link between the creatine kinase system and diseases related to thrombin induced cytoskeletal reorganization, platelet aggregation, inflammatory processes, endothelial cell contraction and related cardiovascular and CNS disorders.
The present invention is directed to a new method of use for creatine compounds such as creatine kinase inhibitors, and more particularly, cyclocreatine and homocyclocreatine, to inhibit thrombin-induced cytoskeletal reorganization, platelet aggregation, inflammatory processes, endothelial cell contraction and related cardiovascular and CNS disorders.
Because these creatine compounds, and particularly creatine kinase inhibitors, modulate intracellular signals of the thrombin receptor PAR-1, creatine compound treatment may be applied in vivo to inhibit pathological platelet aggregation and endothelial cell barrier function.
Cyclocreatine, a competitive inhibitor of creatine kinase, may be taken orally or given intravenously for hypercoaguable, platelet, inflammatory, cardiovascular or cerebrovascular diseases, disorders and conditions. Other known therapies, involving the administration of non-steroidal anti-inflammatory drugs (NSAIDS) such as aspirin, may cause problems with ulceration and bleeding in the stomach. Cyclocreatine may not have the same side effects associated with NSAIDS and other platelet inhibitors.
In practice of the invention, pharmaceutically effective amounts of creatine compounds are administered to subjects in need thereof to thereby prevent and/or treat diseases and/or pathological conditions such as thrombosis, thrombocytopenia, atherosclerosis, coronary artery disease, unstable angina pectoris, myocardial infarction, stroke, coagulopathies, and transient ischemia attacks.
Creatine compounds can also be used to coat devices such as vascular grafts, stents or valves to be placed in contact with blood, to thereby prevent aggregation on the device.